Novel methods and compositions for treatment of autoimmune diseases

ABSTRACT

The subject invention concerns a method for therapeutically treating a patient afflicted with autoimmune disorders or disease. The subject method comprises administering to a patient an antibody composition that is capable of binding to and inhibiting self-reactive pathogenic antibodies present within the patient. Specifically exemplified is a method for treating systemic lupus erythematosis wherein the antibody composition administered to the patient comprises purified anti-DNA anti-idiotypic antibodies. The subject invention further concerns a method for purifying from pooled human gamma globuli preparations anti-idiotype antibodies useful in the present therapeutic method. The present invention further concerns a purified antibody composition useful in the present therapeutic method.

[0001] This invention was made with government support under NationalInstitutes of Health grant number AR 1382421A1. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

[0002] Autoimmune diseases, such as systemic lupus erythematosus (SLE),myasthenia gravis (MG) and idiopathic thrombocytopenic purpura (ITP),among others, remain clinically important diseases in humans. As thename implies, autoimmune diseases wreak their havoc through the body'sown immune system. While the pathological mechanisms differ betweenindividual types of autoimmune diseases, one general mechanism involvesthe binding of certain antibodies (referred to herein as self-reactiveantibodies or autoantibodies) present in the sera of patients to selfnuclear or cellular antigens.

[0003] SLE has an incidence of about 1 in 700 women between the ages of20 and 60. SLE can affect any organ system and can cause severe tissuedamage. Numerous autoantibodies of differing specificity are present inSLE. SLE patients often produce autoantibodies having anti-DNA, anti-Ro,and anti-platelet specificity and which are capable of initiatingclinical features of the disease, such as glomerulonephritis, arthritis,serositis, complete heart block in newborns, and hematologicabnormalities. These autoantibodies are also possibly related to centralnervous system disturbances. Kidney damage, measured by the amount ofproteinuria in the urine, is one of the most acute areas of damageassociated with pathogenicity in SLE, and accounts for at least 50% ofthe mortality and morbidity of the disease. The presence of antibodiesimmunoreactive with double-stranded native DNA is used as a diagnosticmarker for SLE.

[0004] Antibodies are composed of heavy and light polypeptide chainswhich are joined by disulfide bridges. Antibodies are divided intodifferent classes according to their heavy chain structure; antibodiesbelonging to the same class are referred to as isotypes of each other.In addition, antibodies of a given isotype can be divided into subtypes.Antigenic determinants on antibodies that differ among animals that haveinherited different alleles are referred to as allotopes; antibodiesthat share an allotope are referred to as members of the same allotype.Another type of antigenic determinant present on antibody molecules arethose found primarily in the hypervariable region of the antigen bindingsite of the antibody. These determinants are referred to as idiotopes;antibodies that share an idiotope are referred to as members of the sameidiotype. Idiotypic determinants are controlled by both genetic andantigenic influences. Antibodies having common or shared idiotypesgenerally exhibit the same antigenic specificity. However, antibodiesfrom genetically different individuals which share a common antigenicspecificity may exhibit idiotypic heterogeneity but, in some instances,show a major cross-reactive antigenic determinant. Thus, antibodieswhich bind the same antigen may have distinct idiotypic determinants,but also may share cross-reacting properties.

[0005] Currently, there are no really curative treatments for patientsthat have been diagnosed with SLE. From a practical standpoint,physicians generally employ a number of powerful immunosuppressive drugssuch as high-dose corticosteroids, azathioprine or cyclophosphamide—manyof which have potentially harmful side effects to the patients beingtreated. In addition, these immunosuppressive drugs interfere with theperson's ability to produce all antibodies, not just the self-reactiveanti-DNA antibodies. Immunosuppressants also weaken the body's defenseagainst other potential pathogens thereby making the patient extremelysusceptible to infection and other potentially fatal diseases, such ascancer. In some of these instances, the side effects of currenttreatment modalities can be fatal.

[0006] One method of treatment for SLE, described in Diamond et al.(U.S. Pat No. 4,690,905), consists of generating monoclonal antibodiesagainst anti-DNA antibodies (the monoclonal antibodies being referred totherein as anti-idiotypic antibodies) and then using theseanti-idiotypic antibodies to remove the pathogenic anti-DNA antibodiesfrom the patient's system. However, there are several drawbacks to thisapproach. For example, the removal of large quantities of blood fortreatment can be a dangerous, complicated process. Essentially, blood isremoved from a patient, treated to remove the anti-DNA antibodies, andthen the treated blood returned to the patient. Such a removal techniquewould be similar to that used for hemodialysis, i.e., via an arterialpassage. This type of treatment would be inconvenient (a qualifiedprofessional would be required to conduct treatment regularly),expensive, painful, and in some instances might subject the patient to arisk of infection and/or hemorrhaging, as well as depletion of effectiveblood volume inducing circulatory collapse, acute left ventricularfailure or acute renal failure. One treatment session may take hours tocomplete. It also could present certain other risks: heart failurecaused by the rapid transfer of blood, blood loss, acute kidney failuredue to temporary major depletion of effective circulating plasma volume,and/or the possible spreading of dangerous diseases such as HIV,hepatitis B, and hepatitis C. The therapeutic method of the presentinvention avoids these problems. It merely requires an injection, orother equivalent mode of administration, of an antibody composition tothe patient.

[0007] High dose intravenous immune globulin (IVIG) infusions have alsobeen used in treating certain autoimmune diseases. Previous studies haveindicated that IVIG may contain anti-idiotype activity against anti-DNAantibodies, as well as many other autoantibodies (Jordan, S. C., 1989;Silvestris et al., 1994; Mouthon et al., 1996; Silvestris et al., 1996).The effects of IVIG infusions are apparently related to changes in therepertoire of autoantibodies expressed in the patient. This modulationof pathogenic Id antibodies is thought to depend on their specificinteraction with the regulatory anti-idiotype molecules that occurnaturally in healthy donors. Production of anti-idiotypic antibodiesinhibiting the potentially harmful autoimmune repertoire may result fromactivation of the Id network committed to controlling the secretion ofnatural autoantibodies by CD5-positive B cells.

[0008] Up until the present time, treatment of SLE with IVIG hasprovided mixed results, including both resolution of lupus nephritis(Akashi et al., 1990), and in a few instances, exacerbation ofproteinuria and kidney damage (Jordan et al., 1989). The cause of thisincrease is not clear but it is believed that there is increasedglomerular deposition of immune-complexed, polyreactive, non-Id-specificIgG antibodies.

[0009] As can be understood from the above, although there are severaltreatments for autoimmune disease such as systemic lupus erythematosus,all possess serious disadvantages. Thus, persons afflicted with SLE whoshow clinical evidence for SLE nephritis need a cost-efficient and safetreatment that will help prevent or ameliorate the tissue damage thatleads ultimately to kidney failure and the need for chronic hemodialysisand/or renal transplantation caused by their condition.

BRIEF SUMMARY OF THE INVENTION

[0010] The subject invention concerns novel compositions and methods forthe treatment of antibody-based autoimmune diseases, and in particular,SLE. One aspect of the present invention concerns a therapeutic methodfor treating patients suffering from, or predisposed to, autoimmunedisorders such as SLE nephritis. The method comprises administering ananti-idiotypic antibody composition to a patient afflicted with anautoimmune disease, wherein the anti-idiotypic antibodies selectivelyimmunoreact with autoantibodies bearing the appropriate idiotype,thereby inhibiting the autoantibodies and their destructive autoimmuneresponses without inducing generalized immunosuppression. The novelanti-idiotypic antibody compositions, prepared in accordance with theprocedures of the subject invention, comprise anti-idiotypic antibodieshaving specificity for pathogenic self-reactive antibodies in apatient's body, thereby modulating the potential of the self-reactiveantibodies to form immune complexes with self antigens and cause harm tonormal cells and tissues, particularly within the patient's kidneyfiltering systems. One embodiment of the present invention is a methodfor treating SLE using anti-DNA anti-idiotypic antibody compositionsprepared from pooled human intravenous gamma globulin (IVIG). Thepotential risks and negative side effects associated with other currentautoimmune disease therapies are avoided with the present method.

[0011] Another aspect of the subject invention concerns novelcompositions comprising purified human anti-idiotypic antibodies whichhave binding specificity for self-reactive antibodies that areassociated with clinical pathogenesis of certain autoimmune diseases.The antibody compositions are capable of modulating the deleteriouseffect of the self-reactive autoantibodies on cells and tissues of theaffected patient. Specifically exemplified in the present invention is apurified anti-DNA anti-idiotypic antibody composition that can be usedto treat patients afflicted with SLE nephritis.

[0012] In a further aspect, the subject invention is directed toward amethod of producing the novel anti-idiotypic antibody compositions whichcan be used in the therapeutic method of the present invention. Thesubject antibodies can be produced by adsorbing pooled normal humangamma globulin with a solid phase substrate having antibody moleculesattached thereto that selectively bind with anti-idiotypic antibodiespresent in the human gamma globulin, whereby an antibody/anti-idantibody complex is formed on the solid phase. The anti-idiotypicantibodies from the intravenous gamma globulin preparations are theneluted from the solid phase substrate. The procedure allows for theisolation and enrichment of anti-idiotypic antibodies from the otherantibodies present in the pooled gamma globulin preparation.

BRIEF SUMMARY OF FIGURES

[0013] FIGS. 1A-1D show a series of competitive inhibition experimentsin which biotinylated SLE affinity-purified anti-DNA IgG antibodies(APAD) was pre-incubated with either flow-through (FT) or eluates (E)from anti-DNA Id(+) monoclonal antibody-coupled affinity columns. Abroad nanomolar range of concentrations of inhibiting column eluates orflow-through fractions were then tested with the individual biotinylatedSLE APAD preparations. FIGS. 1A and 1B show only slight to moderateinhibition (25-50%) of SLE Pop and Bea APAD by anti-DNA Id(+)-columneluates, and no inhibition by column flow-through fractions. In FIGS. 1Cand 1D major inhibition (70-100% of SLE APAD McK and Bel was recordedwith anti-DNA Id(+)-column eluates. Positive control inhibition of SLEAPAD binding by DNA is shown with the curves marked DNA.

[0014]FIG. 2 shows a diagrammatic representation of theidiotypic/anti-idiotypic assay system. dsDNA was coated on an ELISAplate and biotinylated affinity purified SLE IgG APAD was pre-incubatedwith a broad range of concentrations of anti-DNA Id(+) column eluate(dotted line antibodies). However, since anti-DNA Id(+) column eluateantibodies themselves have anti-DNA reactivity, apparent inhibitionshown by a single * might be observed if anti-DNA Id(+) column eluatestake up a significant fraction of the antigenic sites on ELISA plate DNA(double asteriks**).

[0015]FIG. 3 shows ELISA activities of anti-DNA Id(+) column eluates (E)and flow-through fractions (FT) with SIE IgG F(ab′)₂ as well asspecifically purified F(ab′)₂ fragments of affinity purified SLE IgGanti-DNA F(ab′)₂ (APAD F(ab′)₂) in comparison to reaction with normalsubjects' IgG F(ab′)₂. Much stronger reactions were recorded with bothwhole SLE F(ab′)₂ and affinity purified F(ab′)₂ of SLE anti-DNA andanti-DNA Id(+) column eluates than with column flow-through fractions.No ELISA reaction was noted with IgG F(ab′)₂ from 10 normal subjects.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The subject invention concerns a novel therapeutic method thatcan be used to beneficially treat persons afflicted with autoimmunedisorders. The treatment procedures of the subject invention representan important advance in the care of patients suffering from, orpredisposed to, autoimmune disorders, such as SLE nephritis. Thesedisorders are a disparate collection of human diseases that have incommon the failure of the immune system to recognize a tissue, organ orcellular component of the body as self. As the result of thesepathological processes, a person's own cells and tissues are targetedfor attack by their immune system. Specifically exemplified herein is atreatment procedure to alleviate the destructive effects of SLE on thekidney. Other diseases that can be treated according to the presentinvention include, for example, myasthenia gravis, idiopathicthrombocytopenic purpura, and HIV infection.

[0017] As used herein, the term “anti-DNA antibodies” refers toantibodies that have specificity for double-stranded or native DNA.

[0018] As used herein, the term “anti-DNA idiotype (+)” and “anti-DNAId(+)” refer to anti-DNA antibodies that have been typed positive forthe expression of idiotypic determinants associated with anti-DNAantibodies.

[0019] As used herein, the term “anti-DNA anti-idiotypic antibodies”refers to anti-idiotypic antibodies that have specificity for idiotypicdeterminants associated with anti-DNA antibodies.

[0020] The present therapeutic method comprises administering to apatient in need of treatment an effective amount of an anti-idiotypicantibody composition, or antigen binding fragments thereof, which canbind to and inhibit self-reactive autoantibodies present in the patient.One embodiment of the therapeutic method of the present invention totreat persons diagnosed with, or predisposed to, SLE utilizes anti-DNAanti-idiotypic antibodies, or antigen binding fragments thereof,produced from pooled gamma globulin of normal individuals. The methodcomprises administering to the patient an effective amount of ananti-DNA anti-idiotypic antibody composition that has bindingspecificity for pathogenic self-reactive anti-DNA autoantibodiesproduced by and present in the patient's body. In a preferredembodiment, the anti-idiotypic antibody composition comprises humanantibodies.

[0021] The anti-DNA anti-idiotypic antibody composition is preferablyadministered to a person intravenously. Therapeutic application of theantibody composition can be accomplished by any suitable therapeuticmethod and technique presently or prospectively known to those skilledin the art. The anti-DNA anti-idiotypic antibody composition can beadministered in any suitable solution, such as, for example, a dextrosesaline solution. The amount of antibody composition to be administered,as well as the number of treatments a patient receives, can bedetermined by clinicians skilled in the art. Typically, a patient willreceive multiple treatments over time. Materials used in the presenttreatment method can be sterilized according to conventional means.

[0022] Administration of human-specific anti-idiotypic antibody to SLEpatients may be able to produce a lasting or prolonged down-modulatingeffect on the hyperactive immune reactions present in the SLE patientsimmune system. The anti-DNA anti-idiotypic antibodies interfere with theability of self-reactive anti-DNA antibodies to immunoreact with, andthereby damage, a person's own tissue and cells. The therapeuticadministration of anti-DNA anti-idiotypic antibodies results in asignificant decrease in the amount of anti-DNA antibodies present in thebody of a SLE patient, and reduces the amount of tissue damage in thekidneys.

[0023] Unlike previous treatments for SLE using a general nonspecificpool of commercial gamma globulins, the therapeutic method of thepresent invention is less likely to exacerbate the problem of tissuedamage or trigger an increased autoimmune response in the treatedpatient since the antibody composition administered according to thesubject invention has been treated in a manner such that substantiallyonly antibodies that immunoreact with idiotypes present on anti-DNAantibodies remain in the therapeutic composition. Thus, the method ofthe subject invention overcomes the problems associated with previoustreatments, e.g., tissue damage caused by glomerular deposition ofimmune-complexed, polyreactive, non-anti-DNA Id-specificimmunoglobulins.

[0024] The mechanism of action of the subject method is highly selectiveand only counteracts the adverse effects of destructive antibodiesalready present in the body of the SLE-afflicted patient. No othercomponent of the immune system, albeit humoral or cellular, isdisrupted. Thus, the patient's immune system is not significantlyweakened, unlike those treatment methods where immunosuppressant drugsare used to treat certain autoimmune disorders.

[0025] The present invention also concerns a method for producing acomposition that is highly enriched for anti-idiotypic antibodies orantigen binding fragments thereof, from a pool of gamma globulin,whereby the anti-idiotypic antibodies are reactive with idiotypespresent on antibodies that are associated with certain autoimmunedisorders. In a preferred embodiment, the subject method comprisescontacting pooled, normal human gamma globulin with a solid phasesubstrate comprising antibodies that express idiotypic determinants ofDNA-reactive antibodies (Le, anti-DNA Id(+)), resulting in the formationof an idiotypic/anti-idiotypic antibody complex on the solid phasesubstrate, and then eluting the adsorbed anti-DNA anti-idiotypicantibodies from the substrate. The adsorbed antibodies can be elutedusing methods and materials known in the art, such as, for example, alow pH buffer. The substrate having the adsorbed antibodies presentthereon may be washed with a suitable wash buffer prior to elution.

[0026] The solid phase substrate can include, but is not limited to, anystructural component commonly known in the art that is suitable for suchpurpose, such as DEAE, Sepharose, glass beads, PVDF, nitrocellulose andplastics such as polystrene. The molecules expressing the idiotypicmarkers of interest can be coupled to the solid phase substrate by meansknown in the art In a preferred embodiment, anti-DNA antibodies coupledto the support matrix comprise human IgG that possess at least one ofthe following idiotypic markers: 16/6, F4, 3I, and 8.12. Other anti-DNAantibodies expressing idiotype markers are known in the art and can beused in accordance with the subject invention.

[0027] Idiotypic structural and conformational determinants associatedwith a self-reactive autoantibody, such as, for example, anti-DNAantibodies, for use with the present invention can also be preparedusing hydropathic analysis of primary variable region amino acidsequences of the antibodies, followed by construction of syntheticpeptides capable of duplicating these idiotypic determinants (Blalock,1990; Maier et al., 1994). These synthetic peptides can also be coupledto a solid phase substrate and used to purify anti-idiotypic antibodiesfrom pooled gamma globulin or other fluids. Accordingly, these peptidesare contemplated within the scope of the present methods.

[0028] The subject invention further concerns novel antibodycompositions that can be used to treat patients suffering fromautoimmune disorders, such as SLE, according to the therapeutic methodsdescribed herein. An antibody composition of the invention comprisesanti-idiotypic antibodies that selectively bind to antibodies bearingidiotypes associated with pathogenic self-reactive autoantibodies of thepatient afflicted with the autoimmune disorder. Preferably, theanti-idiotypic antibodies are isolated and enriched from a pool ofnormal human gamma globulin. In a preferred embodiment useful fortreating SLE, the antibody composition is composed of purified anti-DNAanti-idiotypic antibodies which bind to self-reactive anti-DNAantibodies. Preferably, the anti-DNA anti-idiotypic antibodies bindspecifically to those antibodies that are specific for nativedouble-stranded DNA. The anti-idiotypic antibody compositionspecifically exemplified herein has binding specificity for thefollowing human idiotypic markers present on anti-DNA antibodies: 16/6,F4, 3I, and 8.12. However, antibodies with specificity for idiotypemarkers of anti-DNA antibodies other than those specifically exemplifiedherein can be prepared and isolated by the ordinarily skilled artisanusing the teachings contained herein, and are contemplated within thescope of the present invention.

[0029] The antibody composition of the present invention can also beused to detect the presence of self-reactive autoantibodies, such asanti-DNA antibodies, in a sample, such as a biological fluid, usingmethods well known in the art. For example, the antibody composition ofthe invention can be used in radioimmunoassays (RIA) and enzyme-linkedimmunosorbet assays (ELISA). Typically, the subject antibody compositionis contacted with a sample suspected of having self-reactiveautoantibody therein that is immunoreacts with the subject antibody.After contacting the sample and allowing antibody binding to occur,bound antibody is detected using standard techniques. The amount ofautoantibody present in the sample can be quantified using methods wellknown in the art.

[0030] Also included within the scope of the present invention areantigen binding fragments of whole antibody, wherein the fragmentsretain the binding specificity of the whole antibody molecule. Thesebinding fragments include, for example, Fab, F(ab′)₂, and Fv fragments.Binding fragments can be obtained using conventional techniques, such asproteolytic digestion of antibody by papsin or pepsin, or throughstandard genetic engineering techniques that are well known in the art.

[0031] The subject invention also concerns kits comprising in one ormore containers a purified anti-idiotypic antibody composition that hasbinding specificity for self-reactive autoantibodies. In a preferredembodiment, the anti-idiotypic antibody composition of the kit compriseshuman anti-DNA anti-idiotypic antibodies.

Materials and Methods Preparation of Anti-DNA Idiotype Antibody CoupledSubstrate

[0032] Three hundred human IgG myeloma proteins were typed for fourdifferent anti-DNA idiotypic markers (16/6, F4, 3I, and 8.12) aspreviously described (Williams, Jr. et al., 1994). Originally, all 300IgG myelomas were selected on the basis of their cationicelectrophoretic behavior because of the known proclivity of naturallyoccurring anti-DNA reactivity in human SLE and murine SLE models to beassociated with cationic IgG molecules (Gauthier et al, 1982; Ebling etal., 1980; Gallo et al., 1981). A panel of these human IgG myelomaspossessing at least one or several of the four previously characterizedanti-DNA idiotypic markers referred to herein as 16/6, F4, 3I, and 8.12(Shoenfeld et al., 1989; Halpern et al., 1984; Davidson et al., 1989;Davidson et al., 1990) were isolated by diethylaminoethyl cellulose(DEA) chromatography, cross absorbed with insoluble Sepharose 4Bimmunoabsorbents linked to opposite light chain type goat anti-humanKappa or lambda chain antibodies by cyanogen bromide coupling (Williams,Jr. et al., 1994), and then linked to Sepharose 4B to provide animmunoabsorbent column representing human IgG molecules bearing antigenssimilar to or encompassing parts of the respective anti-DNA 16/6, F4,3I, and 8.12 idiotypic markers. 10 mg of anti-DNA idiotype(+) myelomaprotein was used per gram of Sepharose 4B to couple the myeloma proteinsto the Sepharose solid phase substrate. After subsequent extensivewashings of the Sepharose 4B coupled with the anti-DNA idiotype(+)myelomas (Sepharose/anti-DNA idiotype (+) column), no human gammaglobulins were detected in column washings using a sensitive ELISAassay.

[0033] Several controls were included in the series of experimentsinvolving the Sepharose/anti-DNA idiotype(+) column. Two additionalSepharose/myeloma columns were prepared as controls. One columnconsisted of four cationic human IgG myelomas that lacked intrinsicanti-DNA activity and that also lacked positive reactions for the fouranti-DNA idiotypes markers (F4, 3I, 16/6, and 8.12). A second controlcolumn consisted of four strongly anionic IgG myelomas, which were alsonegative by ELISA for anti-DNA antibody activity and negative for eachof the anti-DNA idiotype markers. Pooled human intravenous gammaglobulin preparations were adsorbed and eluted from these controlSepharose/myeloma columns in parallel.

[0034] Three different preparations of intravenous gamma globulins wereselected for study and are identified herein as lots P-3, L-2, and M-1.Each gamma globulin lot represented highly purified human IgG from anestimated pool of 25,000 to 35,000 individual donors. The P-3, L-2, andM-1 lots were chosen for study since they respectively representedrelatively high (O.D. 1.760), medium (O.D. 0.396), and very low (O.D.0.017) levels of generic anti-idiotype IgG anti-F(ab′)₂ (Williams, Jr.et al., 1995a; Williams, Jr. et al., 1995b). Gamma globulin from lotP-3, L-2, or M-1 was applied at room temperature to a Sepharose/humananti-DNA idiotype (+) myeloma column as a solution of 10-20 mg/mlprotein in 0.1 M phosphate-buffered saline (pH 7.4) (PBS) and allowed toadsorb to the column at room temperature for several minutes beforewashing the columns using 0.1 M PBS. The column was then eluted with 0.1M pH 2.5 glycine-saline buffer and the eluates immediately brought toneutral pH (about 7.4) with Tris. Subsequent testing of low pHglycine-saline eluted columns using chaotropic buffers (thiocyanate orcacodylate) did not produce any additional elution of column-adherentIgG, indicating that the glycine-saline elution procedure was relativelyefficient.

[0035] After the gamma globulin-adsorbed Sepharose/anti-DNA idiotype(+)myeloma columns were eluted and the eluates brought to neutral pH, alleluates were adjusted to a protein concentration of 5 mg/ml and testedfor their ability to inhibit binding of affinity purified biotinylatedanti-DNA IgG antibodies (APAD) obtained from a representative panel ofpatients with active SLE. Affinity purified anti-DNA antibody from fourpatients with active SLE nephritis (established by biopsy) and fourother patients with active severe central nervous system (CNS)involvement and high anti-DNA antibody titers were chosen forevaluation. The four SLE patients with CNS involvement showed noclinical evidence for concomitant renal disease (normal urinalysis andnormal serum creatinine and BUN). Affinity purified anti-DNA IgG anybodywas prepared from the serum or plasma of SLE patients using DEAEcellulose chromatography (0.01 sodium, potassium phosphate buffer pH7.0) to purify the IgG fraction from the serum or plasma. The IgG wasthen concentrated to about 5-10 mg/ml and applied to a DNA-coupledSepharose affinity column at neutral pH (pH 7.4). After thorough washingof the affinity column, adherent IgG that had bound to the DNA waseluted at pH 25 with 0.1 M glycine-saline buffer. Eluates wereimmediately brought to neutral pH 7.4 with Tris. The purified anti-DNAIgG preparations were then biotinylated using standard procedure asdescribed in (Bayer et al., 1980). Biotinylated affinity purifiedanti-DNA IgG antibody that immunoreacted with DNA by ELISA was used tomeasure anti-DNA antibody binding to DNA. ELISA assays of affinitypurified anti-DNA IgG binding to DNA on the ELISA plate before and afterbiotinylation showed that the biotinylation procedure did not affect theanti-DNA reactivity of the original affinity purified anti-DNA IgG.Inhibition of biotinylated SLE affinity purified anti-DNA antibodyreacting with DNA on the ELISA plates was estimated by preincubation ofa standard amount of SLE biotinylated affinity purified anti-DNAantibody with either the anti-DNA idiotype(+) myeloma column eluates (E)or flow-through (FT) materials at room temperature for 30 minutes. Therespective eluates and flow-through fractions were tested for inhibitionof affinity purified anti-DNA antibody reacting with DNA by ELISA usinga broad nanomolar concentration range of either anti-DNA idiotype(+)column eluate or flow-through fractions. Biotinylated SLE affinitypurified anti-DNA antibody from nephritis or cerebritis patients wastested using equal amounts of anti-DNA activity as initially tested inthe ELISA assays.

[0036] Assays of Anti-DNA Idiotype Myeloma Column Eluates andFlow-Through Fractions for Anti-DNA and Anti-F(ab′), Specificities

[0037] All anti-DNA idiotype(+) myeloma affinity column eluates andflow-through fractions prepared from pooled human gamma globulinpreparations were tested for both anti-DNA as well as anti-F(ab′)₂,anti-Sm and anti-RNP activity using ELISA assays as previously described(Silvestris et al., 1984; Williams, Jr. et al, 1995a; Williams, Jr. etal., 1995b; Williams, Jr. et al., 1995c). The anti-DNA ELISA assayemployed showed that liquid phase DNA inhibited the binding ofantidouble stranded DNA to the plates. Liquid phase inhibition ofanti-Sm or anti-Sm/RNP was demonstrated for Sm or Sm/RNP antigen,respectively, as well. In addition, column eluates and flow-throughmaterials were also tested for conventional rheumatoid factor (RE)activity using Fc or rabbit IgG coated on ELISA plates at 5 mg/ml, anddeveloped with peroxidase-conjugated goat F(ab′)₂ anti-human light chaindeveloping reagent Assays of ELISA reactivity with Sm and RNP antigen(Immunovision, Springdale, Ark.) were performed as previously described(Williams, Jr. et al, 1995C).

Measurement of Antibody Affinity

[0038] Individual antibody population affinities were also measuredusing an ELISA inhibition method originally described by Devey et al.(1988) with proper attention to pH effects as emphasized by Goldblatt etal. (1993). This method employs an ELISA assay in which diethylamine(DEA) is employed as a chaotropic agent by which functional affinitiesof antibodies to complex antigens can be determined and compared. Themethod is based on determining the leftward shift of a dose-responsecurve for antibody reacting with antigen coated on the ELISA plate afterintroduction of DEA at a pre-established optimal concentration. Affinitywas expressed as the mean shift (log₁₀), and the lower the value, thehigher was the actual functional affinity.

[0039] Following are examples which illustrate materials, methods andprocedures, including the best mode, for practicing the invention. Theseexamples are for illustrative purposes only and are not to be construedas limiting.

EXAMPLE 1 Preparation of Anti-DNA Anti-Idiotypic Antibody Composition

[0040] Three different intravenous gamma globulin preparations (P3, L-2,and M-1) were adsorbed to the Sepharose/anti-DNA idiotype (+) humanmyeloma affinity columns as described in the material and methodssection. Most of the gamma globulin in the solution did not adsorb tothe column but passed through the column and remained in theflow-through fractions. However, a small proportion (0.04-0.9%) of theoriginal gamma globulin did adsorb to the affinity columns. When theadsorbed fraction was subsequently eluted from the column at low pHtests for anti-DNA anti-idiotype activity in such myeloma column eluatesoften showed potent inhibition of biotinylated SLE affinity purifiedanti-DNA antibody reacting with DNA on the ELISA plate. Thus, in severalinstances biotinylated affinity purified anti-DNA antibody from SLEpatients reacting with DNA by ELISA showed complete inhibition at highernanomolar concentrations of the anti-DNA idiotype(+) column eluates. Bycontrast, in no instances did column flow-through fractions showinhibition of affinity purified anti-DNA antibody binding to DNA asmeasured by ELISA. Although gamma globulin lots with high baselineanti-F(ab′)₂ activity frequently showed anti-DNA idiotype(+) columneluates with strong blocking activity for several SLE affinity purifiedanti-DNA IgG preparations, no clear correlation between anti-F(ab′)₂activity and column eluted anti-DNA anti-idiotype (anti-combining site)activity was observed. Thus, in some instances eluates from gammaglobulin lots with low anti-F(ab′)₂ activity showed strong paratope(combining site) inhibition of affinity purified anti-DNA antibodiesfrom SLE patients. Examples of these results are shown in FIGS. 1A and1B. A summary of the affinity purified anti-DNA antibody inhibitionresults obtained with eight different SLE patients is shown in Table 1.TABLE 1 Relative degrees of Inhibition of Biotinylated SLE APAD reactingwith DNA on ELISA plates by a broad nanomolar range of 3 differentpreparations of IVIG (Baxter) Intermediate anti- High anti-F(ab′)₂F(ab′)₂ Low anti-F(ab′)₂ SLE APAD* P3 L2 M-1 Tested Nanomolar⁺ NanomolarNanomolar Nephritis 10 10² 10³ 10 10² 10³ 10 10² 10³ McK 25** 45 80 1654 75 20 55 70 Lar 15 35 80 20 40 95 42 50 100 McN 0 10 45 26 50 87 8 2078 War 12 30 58 0 4 25 0 0 38 Mean 13 30 66 15 37 70 18 31 72 CNS 10 10²10³ 10 10² 10³ 10 10² 10³ Pop 0 0 15 16 34 66 0 0 25 Bel 20 47 90 0 2870 0 4 58 Bea 5 18 42 0 2 20 0 2 20 Mor 0 10 50 4 8 45 0 0 38 Mean 6 1949 5 18 50 0 2 35

[0041] In general, biotinylated affinity purified anti-DNA antibody frompatients with active SLE nephritis showed more inhibition by anti-DNAidiotype(+) myeloma column eluates than was observed with biotinylatedaffinity purified anti-DNA antibody isolated from the four patients withactive central nervous system lupus (Table 1). Parallel control studieswith cationic and anionic myelome-affinity columns all withoutexpression of any of the four major anti-DNA idiotype markers (16/6, F4,3I or 8.12) showed that quantitative amounts of gamma globulin whichactually adsorbed to such columns were less than one quarter to oneeighth the amounts adsorbed from the same lots of P-3, L-2, or M-1 gammaglobulin. Moreover, none of the eluates of flow through fractions fromsuch control anti-DNA idiotype-negative cationic or anionic myelomaaffinity columns showed any significant inhibition in the biotinylatedSLE affinity purified anti-DNA antibody experiments similar to thoseillustrated in FIGS. 1A-D or in Table 1. These results suggest that inorder to concentrate functional anti-DNA anti-idiotype reactivity fromgamma globulin preparations capable of blocking individual SLE anti-DNAantibody it was necessary to adsorb such preparations on cationicanti-DNA idiotypic(+) myeloma immunoadsorbents rather than merely pooledcationic myelomas alone.

EXAMPLE 2 Anti-DNA Antibody, and Anti-F(ab′)₂, Anti-Sm and Anti-RNPAntibody Activity in Anti-DNA Idiotypic(+) Column Eluates andFlow-Through Fractions

[0042] Column eluates of gamma globulin which had adhered toSepharose/anti-DNA idiotype(+) myeloma affinity columns were studied inparallel with flow-through fractions for intrinsic anti-DNA as well asanti-F(ab′)₂ and anti-Sm or anti-Sm/RNP activity by ELISA. Thus, myelomaanti-DNA idiotype(+) column eluates from all three lots of gammaglobulin (P-3, L-2, and M-1) showed considerable relative concentrationof anti-DNA activity compared with activity in the starting lots ofgamma globulin studied at the same IgG concentrations (see Table 2).TABLE 2 ELISA reactions of intravenous gamma globulin preparations P3and L2 before and after adsorption to anti-DNA Id(+) human IgG myelomacolumn Intravenous gamma ELISA Anti- globulin preparation Anti-DNAF(ab′)₂ Anti-Sm Anti-RNP IVIG P3 untreated 0.521* 1.760+ 0.235+ 0.276+IVIG P3 over anti-Id + 0.193 0.105 0.287 0.353 column (flow throughfraction) P3 Eluate from anti-Id + 1.704 1.400 1.054 2.194 human myelomacolumn IVIG L2 0.585* 0.396 0.161 0.215 IVIG L2 over anti-Id + 0.6160.064 0.148 0.214 column (flow through fraction) L2 Eluate fromanti-Id + 0.937 1.049 0.547 0.719 column

[0043] Gamma globulin from lot P3 showed 0.521 OD anti-DNA reactivitycomposed with only 0.193 OD for the flow-through fraction. The P3 low pHeluate produced a 1.704 OD in the anti-DNA ELISA assay. In parallel, theunadsorbed gamma globulin lot P3 showed relatively high anti-F(ab′)₂with little residual anti-F(ab′)₂ in flow-through fractions, and againhigh anti-F(ab′)₂ activity in the anti-DNA idiotype(+) column eluate.Although baseline gamma globulin lot P3 showed low ELISA values foranti-Sm and anti-RNP activity, low pH anti-DNA idiotype(+) columneluates showed marked relative concentration of each of these antibodyactivities as well. A similar profile was noted with gamma globulin M-1before and after adsorption on the anti-DNA idiotype(+) myeloma affinitycolumn.

[0044] When similar adsorption studies of the same three gamma globulinpreparations were performed using the anti-DNA idiotypic-negativecationic and anionic myeloma affinity columns, no marked relativeincrements in anti-DNA or anti-F(ab′)₂, anti-Sm, or Sm/RNP were recordedin column eluates with respect to flow-through fractions or originalstarting material. These findings confirmed the fundamental importanceof using cationic myeloma proteins which were positive for severalcommon anti-DNA idiotypic markers.

[0045] Since gamma globulin anti-DNA idiotype(+) column eluates could bedemonstrated to contain a relative increase in anti-DNA reactivity overand above that present in the starting gamma globulin preparations, itseemed possible that preincubation of test SLE biotinylated affinitypurified anti-DNA antibody with anti-DNA idiotype(+) column eluateswhich resulted in inhibition of biotinylated SLE affinity purifiedanti-DNA antibody binding to DNA as determined by the ELISA mightreflect competition for DNA binding between the biotinylated SLEaffinity purified anti-DNA antibody and the anti-DNA idiotype(+) columneluate (see FIG. 2). In order to examine this question directly,anti-DNA idiotype(+) column eluates and flow-through fractions werecompared for their intrinsic binding activity against IgG F(ab′),fragments of normal subjects and SLE patients' IgG in parallel withtheir binding for F(ab′), fragments of SLE affinity purified anti-DNAantibody. As shown in FIG. 3, these experiments clearly showed thatanti-DNA idiotype(+) column eluates (but not flow-through fractions)showed marked relative binding to both IgG F(ab′) from SLE patients andF(ab′)₂ fragments of SLE affinity purified anti-DNA antibody, but verylittle binding to F(ab′)₂ from individual normal subjects. FIG. 3 showsthat in one instance, eluates showed stronger reactivity for F(ab′)₂ ofSLE affinity purified anti-DNA antibody than merely whole SLE IgG F(ab′)fragments. Such results support apparent marked anti-DNA idiotypespecificity within eluate fractions for antigenic determinants onvariable regions (and presumably combining sites) of specificallypurified SLE anti-DNA antibodies.

[0046] Relative affinities of anti-DNA idiotypic(+) column eluates forDNA were measured using the DEA dissociation ELISA inhibition method(Devey et al., 1988) and compared to relative affinities of a broadpanel of affinity purified anti-DNA IgG antibodies from SLE patients.Representative data are shown in Table 3. TABLE 3 Relative functionalaffinity of anti-DNA Id(+) myeloma affinity column eluates for DNA inparallel with anti-DNA affinity of SLE patients APAD Log₁₀ conc with 25mM DEA Log₁₀ conc at at 50% Max Samples Tested 50% Max OD at 50% MaxLog₁₀ Shift IVIG P-3 Id-column 1.88 1.13 0.75* eluate IVIG L-2 Id column1.93 1.21 0.72 eluate IVIG M-1 Id column 1.52 0.54 0.98 eluate SLE IgGAPAD McN** 1.43 1.02 0.41 SLE IgG APAD Ag 1.53 1.08 0.45 SLE IgG APAD Op1.76 1.67 0.09 SLE IgG APAD Gio 1.51 1.23 0.15 SLE IgG APAD Dan 1.511.32 0.37

[0047] In all instances, affinity for DNA was hiether in affinitypurified anti-DNA antibody preparations from SLE patients than wasrecorded with anti-DNA idiotypic(+) column eluates adsorbed with lotsP-3, L-2, or M-1. These findings indicate a much higher DNA affinitywithin SLE patients' circulating anti-DNA IgG antibody that with theanti-DNA idiotypic(+) column eluates.

EXAMPLE 3 Treatment and Evaluation of SLE Patients

[0048] Patients with established SLE who meet ARA (American RheumatismAssociation) criteria for definite SLE and who show clearcut clinicaland laboratory evidence of SLE nephritis will be selected for treatmentMost patients will have had their SLE nephritis previously consumed anddocumented with a recent kidney biopsy. Patients will typically receiveintravenous anti-DNA anti-idiotypic antibody preparations prepared asdescribed herein once every six weeks for a total of five treatments. Ateach visit they will have routine urinalysis and blood drawn foranti-nuclear antibody, anti-DNA, and anti-F(ab′)₂ determinations. At thebeginning of the study, and after the third and fifth treatment, 24 hoururines and blood will be collected for 24 hour urine protein excretionand creatinine clearance determination Six weeks following thecompletion of the course of therapy and again three months followingcompletion of the treatment protocol, all patients will be re-evaluatedin clinic with CBC, urine, chemistry profile, ANA, anti-DNA and 24 hoururine for protein and creatinine clearance to re-assess stability oftheir overall condition and renal function.

[0049] Anti-DNA anti-idiotypic antibody compositions can be administeredas 150 mg of IVIG in a running solution of 5% Dextrose in saline over aperiod of 1-2 hours. is is similar to the current recommended protocolof regular IVIG administration where a much larger total amount andconcentration (400 mg/Kg or 28 grams for a 70 Kg individual) is usuallygiven. The usual starting rate of IV administration is 0.5 ml/Kg hr of a5% gamma globulin solution increasing over one hour to 4 ml/Kg/hr. Thesame general protocol for IVIG can be followed with the present method;thus, the total of 150 mg of the anti-DNA anti-idiotypic antibodycomposition can be infused as a 1% solution at 0.5 ml/Kg/hr over thefirst hour and then if no untoward reaction is noted in the patient,gradually the rate of administration can be increased to 4 ml/Kg/hruntil the total dose of 150 mg is given.

[0050] It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims.

References

[0051] Akashi, K. K. Nagasawa, T. Mayimi et al. (1990) “Successfultreatment of refractory systemic lupus erythematosus with intravenousimmuno-globulin” J. Rheumatology 17:375-379.

[0052] Blalock, J. E. (1990) “Complementary of peptides specified by“sense” and “anti-sense” strands of DNA. Trends in Biotechnol 8:140-144.

[0053] Diamond et al., U.S. Pat. No. 4,690,905, issued Sep. 1, 1987.

[0054] Bayer, E. A. and M. Wilchek (1980) “The use of avidin-biotincomplex as a tool in molecular biology methods” Biochem Anal. 26:145.

[0055] Davidson, A., A. Manheimer-Lory, C. Aranow R. Peterson, N.Hannigan, B. Diamond (1990) “Molecular characterization of asomatically-mutated anti-DNA antibody being two systemic lupuserythematosus-related idiotypes” J. Clin. Invest. 85:1401-1409.

[0056] Devey, M. E., K. Bleasdale, S. Lee, S. Rath (1988) “Determinationof the functional affinity of IgG1 and IgG4 antibodies to tetanus toxoidby isotype-specific solid-phase assays” J. Immunol. Method 106:119-125.

[0057] Ebling, F., B. E. Hahn (1980) “Restricted subpopulations ofanti-DNA antibodies in kidney of mice with systemic lupus: comparison ofantibodies in serum and renal eluates” Arthritis Rheum. 23:392-403.

[0058] Gallo, G. R., T. Caulin-Glaser, M. E. Lamm (1981) “Charge ofCirculating immune complexes as a factor in glomerular basement membranelocalization” J. Clin. Invest. 67:1305-1313.

[0059] Gauthier, V. J., M. Mannik, G. E. Striker (1982), “Effect ofcatonized antibodies in performed immune complexes on deposition andpersistence in renal glomeruli” J. Exp. Med. 156:766-777.

[0060] Goldblatt, D., L. vanEtten, F. J. van Milligen, R. C. Aalberse,M. W. Turner (1993) “The role of pH in modified ELISA procedures usedfor the estimation of functional antibody affinity” J. Immunol. Methods166:281-285.

[0061] Jordan, S. C. (1989) “Intravenous gamma globulin therapy insystemic lupus eyhematosus and immune complex disease” Clin. Immunol.Immunopathol. 53:S164-169.

[0062] Maier, C. C., H. N. B. Moseley, S. Zhou, J. N. Whitaker, J. E.Blalock (1994) “Identification of interactive determinants onidiotypic-anti-idiotypic antibodies through comparison of theirhydropathic profiles. Complementary peptides: application in immunologyand as antibody mimetics” ImmunoMethods 5:107-113.

[0063] Mouthon, L., S. V. Kaveri, S. H. Spalter, S. Lacroix-Desmazes, C.Lefranc, R. Desai, M. D. Kazatchkine (1996) “Mechanisms of action ofintravenous immune globulin in immune-mediated diseases” Clin. Exp.Immunol. 104:(suppl. 1)3-9.

[0064] Shoenfeld, Y., H. A. Teplizky, S. Mendlovic, M. Blank, E. Mozes,D. Isenberg (1989) “Short analytical review: the role of the humananti-DNA idiotype 16/6 in autoimmunity” Clin. Inmnunol Immunopathol51:313-325.

[0065] Silvestris, F., A. D. Bankhurst, R. P. Searles, R. C. Williams,Jr. (1984) “Studies of anti-F(ab′)₂ antibodies and possible immunologiccontrol mechanisms in systemic lupus” Arthritis Rheum 27:1387-1396.

[0066] Silvestris, F., P. Cafforio, F. Dammacco (1994) “Pathogenicanti-DNA idiotype-reactive IgG in intravenous immunoglobulinpreparations” Clin. Exp. Immunol. 97:10-25.

[0067] Williams, Jr., R. C., C. C. Malone, G. R. Huffman, F. Silvestris,B. P. Croker, E. Ayoub, S. Massengul (1995a) “Active SLE is associatedwith depletion of the natural generic anti-Id (anti-F(ab′)₂) system” J.Reumatol. 22:1075-1088.

[0068] Williams, Jr., R. C., C. C. Malone, F. Silvestris (1995b) “CDRmolecular localization of possible anti-idiotypic anti-DNA antibodies innormal subjects, SLE patients and SLE first-degree relatives” J. Lab andClin. Med. 126:44-56.

[0069] Williams, Jr., R. C., A. D. Schnrer, C. C. Malone, F. Silvestris,N. Hannigan, Klein-Gitelman, M. Namyst, R. A. Kyle (1994). “Expressionof F4, 8.12, 3I and 16/6 anti-DNA idiotype-related antigens on cationichuman IgG myeloma proteins.” Clinical Immunology & Immunopathol.73:215-223.

[0070] Williams, Jr., R. C., C. C. Malone, K. Cimbanik F. Silvestris, K.H. Roux, L. Strelets (1995c) “Human IgG anti-F(ab′)₂ antibody crossreacts with DNA and other nuclear antigens” Arthritis Rheum 38:730,S275.

I claim:
 1. A method for treating a patient suffering from an autoimmunedisorder, said method comprising administering an effective amount of anantibody composition, wherein said antibody composition comprisesanti-idiotype antibodies, or antigen binding fragments thereof, whichcan bind to and inhibit self-reactive pathogenic autoantibodies presentin the patient.
 2. The method, according to claim 1 , wherein saidautoimmune disorder is characterized by the destruction of patients'cells by autoantibodies.
 3. The method, according to claim 2 , whereinsaid anti-idiotype antibodies are immunoreactive with idiotypicdeterminants present or antibodies associated with an autoimmune diseaseselected from the group comprising systemic lupus erythematosus,masthenia gravis, and idiopathic thrombocytopenic purpura.
 4. Themethod, according to claim 3 , wherein said anti-idiotype antibodies areimmunoreactive with anti-DNA antibodies.
 5. The method, according toclaim 4 , wherein said anti-idiotype antibodies are immunoreactive withidiotypic determinants present on said anti-DNA antibodies, wherein saididiotypic determinants are selected from the group consisting of F4, 3I,8.12, and 16/6.
 6. The method, according to claim 1 , wherein saidantibody composition is administered intravenously.
 7. The method,according to claim 6 , wherein said antibody composition is administeredas a dextrose saline solution.
 8. A method for preparing an antibodycomposition comprising anti-idiotype antibodies, comprising the stepsof: (a) contacting pooled gamma globulin with a solid phase substrate,said substrate comprising an idiotypic determinant of a self-reactivepathogenic autoantibody, wherein said anti-idiotype antibodies presentin said pooled gamma globulin bind to said idiotypic determinant; and(b) eluting said anti-idiotype antibodies bound to said idiotypicdeterminant.
 9. The method, according to claim 8 , wherein saididiotypic determinant is expressed on an antibody coupled to saidsubstrate.
 10. The method according to claim 9 wherein said antibodycoupled to said substrate expresses anti-DNA idiotypic determinantsselected from the group consisting of F4, 3I, 8.12 and 16/6.
 11. Themethod according to claim 8 further comprising washing said substratewith a suitable wash buffer prior to eluting in Step (b).
 12. Themethod, according to claim 8 , wherein said substrate comprises aplurality of idiotypic determinants of self-reactive pathogenicautoantibody.
 13. The method, according to claim 8 , wherein saididiotypic determinant comprises a synthetic peptide that duplicates anidiotypic determinant present on a self-reactive autoantibody.
 14. Anantibody composition, wherein said antibody composition comprisesanti-idiotypic antibodies, or antigen binding fragments thereof, whichcan bind to and inhibit self-reactive pathogenic autoantibodies.
 15. Theantibody composition, according to claim 14 , wherein saidanti-idiotypic antibodies are immunoreactive with idiotypic determinantspresent or antibodies associated with an autoimmune diseased selectedfrom the group comprising systemic lupus erythematosus, mastheniagravis, and idiopathic thrombocytopenic purpura.
 16. The antibodycomposition, according to claim 14 , wherein said anti-idiotypicantibodies are immunoreactive with anti-DNA antibodies.
 17. The antibodycomposition, according to claim 16 , wherein said anti-idiotypicantibodies are immunoreactive with idiotypic determinants present onsaid anti-DNA antibodies, wherein said idiotypic determinants areselected from the group consisting of F4, 3I, 8.12, and 16/6.